Interferon inducing genetically engineered attenuated viruses

ABSTRACT

The present invention relates to genetically engineered attenuated viruses and methods for their production. In particular, the present invention relates to engineering live attenuated viruses which contain a modified NS gene segment. Recombinant DNA techniques can be utilized to engineer site specific mutations into one or more noncoding regions of the viral genome which result in the down-regulation of one or more viral genes. Alternatively, recombinant DNA techniques can be used to engineer a mutation, including but not limited to an insertion, deletion, or substitution of an amino acid residue(s) or an epitope(s) into a coding region of the viral genome so that altered or chimeric viral proteins are expressed by the engineered virus.

This application is a continuation of U.S. application Ser. No.09/631,277, filed Aug. 3, 2000, now abandoned, which is a continuationof U.S. application Ser. No. 09/332,287, filed Jun. 11, 1999, now U.S.Pat. No. 6,468,544, which claims priority benefit of U.S. provisionalapplication Ser. No. 60/089,103, filed Jun. 12, 1998, each of which areincorporated herein by reference in their entirety.

The work reflected in this application was supported, in part, by agrant from the National Institutes of Health, and the Government mayhave certain rights in the invention.

1. INTRODUCTION

The present invention relates to engineering attenuated viruses byaltering a non-coding region or the coding sequence of a viralnonstructural (NS) gene. In particular, the present invention relates toengineering live attenuated influenza viruses which induce interferonand related pathways. The present invention further relates to the useof the attenuated viruses and viral vectors against a broad range ofpathogens and/or antigens, including tumor specific antigens. Thepresent invention also relates to a host-restriction based selectionsystem for the identification of genetically manipulated influenzaviruses. In particular, the present invention relates to a selectionsystem to identify influenza viruses which contain modified NS genesegments.

2. BACKGROUND OF THE INVENTION 2.1. Attenuated Viruses

Inactivated virus vaccines are prepared by “killing” the viral pathogen,e.g., by heat or formalin treatment, so that it is not capable ofreplication. Inactivated vaccines have limited utility because they donot provide long lasting immunity and, therefore, afford limitedprotection. An alternative approach for producing virus vaccinesinvolves the use of attenuated live virus vaccines. Attenuated virusesare capable of replication but are not pathogenic, and, therefore,provide for longer lasting immunity and afford greater protection.However, the conventional methods for producing attenuated virusesinvolve the chance isolation of host range mutants, many of which aretemperature sensitive; e.g., the virus is passaged through unnaturalhosts, and progeny viruses which are immunogenic, yet not pathogenic,are selected.

Recombinant DNA technology and genetic engineering techniques, intheory, would afford a superior approach to producing an attenuatedvirus since specific mutations could be deliberately engineered into theviral genome. However, the genetic alterations required for attenuationof viruses are not known or predictable. In general, the attempts to userecombinant DNA technology to engineer viral vaccines have mostly beendirected to the production of subunit vaccines which contain only theprotein subunits of the pathogen involved in the immune response,expressed in recombinant viral vectors such as vaccinia virus orbaculovirus. More recently, recombinant DNA techniques have beenutilized in an attempt to produce herpes virus deletion mutants orpolioviruses which mimic attenuated viruses found in nature or knownhost range mutants. Until very recently, the negative strand RNA viruseswere not amenable to site-specific manipulation at all, and thus couldnot be genetically engineered.

2.2. The Influenza Virus

Virus families containing enveloped single-stranded RNA of thenegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae) or those having segmentedgenomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). TheOrthomyxoviridae family, described in detail below, and used in theexamples herein, contains only the viruses of influenza, types A, B andC.

The influenza virions consist of an internal ribonucleoprotein core (ahelical nucleocapsid) containing the single-stranded RNA genome, and anouter lipoprotein envelope lined inside by a matrix protein (M). Thesegmented genome of influenza A consists of eight molecules (seven forinfluenza C) of linear, negative polarity, single-stranded RNAs whichencode ten polypeptides, including: the RNA-directed RNA polymeraseproteins (PB2, PB1 and PA) and nucleoprotein (NP) which form thenucleocapsid; the matrix proteins (M1, M2); two surface glycoproteinswhich project from the lipoprotein envelope: hemagglutinin (HA) andneuraminidase (NA); and nonstructural proteins whose function is unknown(NS1 and NS2). Transcription and replication of the genome takes placein the nucleus and assembly occurs via budding on the plasma membrane.The viruses can reassort genes during mixed infections.

Influenza virus adsorbs via HA to sialyloligosaccharides in cellmembrane glycoproteins and glycolipids. Following endocytosis of thevirion, a conformational change in the HA molecule occurs within thecellular endosome which facilitates membrane fusion, thus triggeringuncoating. The nucleocapsid migrates to the nucleus where viral mRNA istranscribed as the essential initial event in infection. Viral mRNA istranscribed by a unique mechanism in which viral endonuclease cleavesthe capped 5′-terminus from cellular heterologous mRNAs which then serveas primers for transcription of viral RNA templates by the viraltranscriptase. Transcripts terminate at sites 15 to 22 bases from theends of their templates, where oligo(U) sequences act as signals for thetemplate-independent addition of poly(A) tracts. Of the eight viral mRNAmolecules so produced, six are monocistronic messages that aretranslated directly into the proteins representing HA, NA, NP and theviral polymerase proteins, PB2, PB1 and PA. The other two transcriptsundergo splicing, each yielding two mRNAs which are translated indifferent reading frames to produce M1, M2, NS1 and NS2. In other words,the eight viral mRNAs code for ten proteins: eight structural and twononstructural. A summary of the genes of the influenza virus and theirprotein products is shown in Table I below.

TABLE I INFLUENZA VIRUS GENOME RNA SEGMENTS AND CODING ASSIGNMENTS^(a)Length_(d) Molecules Length_(b) Encoded (Amino Per Segment (Nucleotides)Polypeptide_(c) Acids) Virion Comments 1 2341 PB2 759 30-60 RNAtranscriptase component; host cell RNA cap binding 2 2341 PB1 757 30-60RNA transcriptase component; initiation of transcription; endonucleaseactivity? 3 2233 PA 716 30-60 RNA transcriptase component; elongation ofmRNA chains? 4 1778 HA 566  500 Hemagglutinin; trimer; envelopeglycoprotein; mediates attachment to cells 5 1565 NP 498 1000Nucleoprotein; associated with RNA; structural component of RNAtranscriptase 6 1413 NA 454  100 Neuraminidase; tetramer; envelopeglycoprotein 7 1027 M₁ 252 3000 Matrix protein; lines inside of envelopeM₂ 96 Structural protein in plasma membrane; spliced mRNA ? ?9Unidentified protein 8 890 NS₁ 230 Nonstructural protein; functionunknown NS₂ 121 Nonstructural protein; function unknown; spliced mRNA^(a)Adapted from R. A. Lamb and P. W. Choppin (1983), Reproduced fromthe Annual Review of Biochemistry, Volume 52, 467-506. ^(b)For A/PR/8/34strain ^(c)Determined by biochemical and genetic approaches^(d)Determined by nucleotide sequence analysis and protein sequencing

The Influenza A genome contains eight segments of single-stranded RNA ofnegative polarity, coding for nine structural and one nonstructuralproteins. The nonstructural protein NS1 is abundant in influenza virusinfected cells, but has not been detected in virions. NS1 is aphosphoprotein found in the nucleus early during infection and also inthe cytoplasm at later times of the viral cycle (King et al., 1975,Virology 64: 378). Studies with temperature-sensitive (ts) influenzamutants carrying lesions in the NS gene suggested that the NS1 proteinis a transcriptional and post-transcriptional regulator of mechanisms bywhich the virus is able to inhibit host cell gene expression and tostimulate viral protein synthesis. Like many other proteins thatregulate post-transcriptional processes, the NS1 protein interacts withspecific RNA sequences and structures. The NS1 protein has been reportedto bind to different RNA species including: vRNA, poly-A, U6 _(sn)RNA,5′ untranslated region as of viral mRNAs and ds RNA (Qiu et al., 1995,Rna 1: 304; Qiu et al., 1994, J. Virol. 68: 2425). Expression of the NS1protein from cDNA in transfected cells has been associated with severaleffects: inhibition of nucleo-cytoplasmic transport of mRNA, inhibitionof pre-mRNA splicing, inhibition of host mRNA polyadenylation andstimulation of translation of viral mRNA (Fortes, et al., 1994, Embo J.13: 704; Enami, K. et al, 1994, J. Virol. 68: 1432 de la Luna, et al.,1995, J. Virol. 69:2427; Lu, Y. et al., 1994, Genes Dev. 8:1817; Park,et. al., 1995, J. Biol Chem. 270, 28433).

3. SUMMARY OF THE INVENTION

The present invention relates to genetically engineered live attenuatedviruses which induce an interferon and related responses. In a preferredembodiment the present invention relates to engineering live attenuatedinfluenza viruses which contain modified NS gene segments. The presentinvention also relates to both segmented and non-segmented virusesgenetically engineered to have an attenuated phenotype and an interferoninducing phenotype, such a phenotype is achieved by targeting the viralgene product which interferes with the cellular interferon response. Theattenuated viruses of the present invention may be engineered byaltering the non-coding region of the NS gene segment that regulatestranscription and/or replication of the viral gene so that it is downregulated. In non-segmented viruses, the down regulation of a viral genecan result in a decrease in the number of infectious virions producedduring replication, so that the virus demonstrates attenuatedcharacteristics. A second approach involves engineering alterations ofthe NS coding region so that the viral protein expressed is altered bythe insertion, deletion or substitution of an amino acid residue or anepitope and an attenuated chimeric virus is produced. This approach maybe applied to a number of different viruses and is advantageously usedto engineer a negative strand RNA virus in which a NS gene product playsa role in regulating the interferon-mediated inhibition of translationof viral proteins.

The present invention is further related to vaccines and methods ofinhibiting viral infection. The attenuated viruses of the presentinvention may be used to protect against viral infection. Asdemonstrated by the evidence presented in the Examples herein, theattenuated viruses of the present invention have anti-viral activitywhen administered prior to infection with wild-type virus, thusdemonstrating the prophylactic utility of the attenuated viruses of thepresent invention.

The present invention is further related to a host-restriction basedselection system for the identification of genetically manipulatedinfluenza viruses. The selection system of the present invention is moreparticularly related to the identification of genetically manipulatedinfluenza viruses which contain modified NS gene segments.

The present invention is based, in part, on the Applicants' surprisingdiscovery that an engineered influenza A virus deleted of the NS1 genewas able to grow in a cell line deficient in type 2 IFN production, butwas undetectable in Madin-Darby canine kidney (MDCK) cells and in theallantoic membrane of embryonated chicken eggs, two conventionalsubstrates for influenza virus. The Applicants' further discovered thatthe infection of human cells with the engineered influenza A virusdeleted of the NS1 gene, but not the wild-type virus, induced highlevels of expression of genes under control of IFN-induced promoter.These results allow for the first time an efficient selection system forinfluenza viruses which contain NS1 mutants, where previously it was notpossible to screen for viruses with an NS1 deleted phenotype.

The attenuated viruses of the invention may advantageously be usedsafely in live virus vaccine formulation. As used herein, the term“attenuated” virus refers to a virus which is infectious but notpathogenic; or an infectious virus which may or may not be pathogenic,but which either produces defective particles during each round ofreplication or produces fewer progeny virions than does thecorresponding wild type virus during replication. Pathogenic viruseswhich are engineered to produce defective particles or a reduced numberof progeny virions are “attenuated” in that even though the virus iscapable of causing disease, the titers of virus obtained in a vaccinatedindividual will provide only subclinical levels of infection.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representations of the NS genes and NS-specific mRNAsof (A) wild-type influenza A/PR/9/34 virus (WT NS) and (B) transfectantdelNS1 influenza virus. Genomic RNA segments are represented as whiteboxes flanked by black squares. The latter represent the non codingregions of the gene. NS-specific mRNAs are also represented. Thin linesat the ends of the mRNAs represent untranslated regions. 5′ capstructures (black circles) and poly(A) tails in the mRNAs are shown. Theopen reading frame of the NS1 protein is represented as a grey box. Thespecific-NEP (Nuclear Export Protein) open reading frame is shown as ahatched box. The NEP mRNA derived from the wild-type NS gene is aspliced product of the NS1 mRNA, as indicated by the V-shaped line.

FIG. 2. RT-PCR analysis of the NS RNA segment of delNS1 transfectantvirus. The NS viral RNA from purified influenza A/PR/8/33 virus (wt) orfrom delNS1 virus (delNS1) was amplified by coupled reversetranscription-PCR using the oligonucleotide primers described in Section6. The PCR products were run on a 2% agarose gel and stained withethidium bromide. The positions of size markers are indicated on theright.

FIG. 3. Protein expression in delNS1 virus-infected (A) Vero cells and(B) MDCK cells. Cells were infected with delNS1 virus at an MOI of 0.02,[³⁵S] labeled at the indicated time points, and total amount of viralproteins was immunoprecipitated using a polyclonal antiserum againstinfluenza virus. Immunoprecipitated products were analyzed by SDS-PAGE.The major structural viral proteins, hemagglutinin (HA), nucleoprotein(NP), neuraminidase (NA) and matrix protein (M1) are indicated by thearrows. Molecular size markers are shown on the left.

FIG. 4. Protein expression in delNS1 virus-infected IFNαR−/− cells.Cells were infected with delNS1 virus at an MOI of 0.02, and [³⁵S]labeled at the indicated time points. As a control, delNS1virus-infected Vero cells were labeled in the same experiment from 8 to10 h postinfection. Total amount of viral proteins wasimmunoprecipitated using a polyclonal antiserum against influenza virus.Immunoprecipitated products were analyzed by SDS-PAGE. The majorstructural viral proteins, hemagglutinin (HA), nucleoprotein (NP),neuraminidase (NA) and matrix protein (M1) are indicated by the arrows.Molecular size markers are shown on the left.

FIG. 5. Induction of transcription from an IFN-stimulated promoter byinfection with delNS1 virus. 293 cells were transfected with plasmidpHISG54-1-CAT encoding the reporter gene CAT under the control of a typeI IFN-stimulated promoter. One day posttransfection, cells weretransfected with 50 μg of dsRNA, or infected with delNS1 virus or withwild-type influenza A/PR/8/34 virus (wt) at the indicated MOIs. One daypostinfection, CAT activity was determined in cell extracts. Thestimulation of CAT activity following the different treatments isindicated.

FIG. 6. Induction of antiviral response in embryonated eggs by delNS1virus. 10-day old embryonated chicken eggs were inoculated with 20,000plaque forming units of delNS1 virus or with PBS (untreated). After 8 hincubation at 37° C., the eggs were reinfected with 10³ pfu of H1N1influenza A/WSN/33 virus (WSN), H1N1 influenza A/RP/8 virus (PR8), H3N2influenza A/X-31 virus (X-31), influenza B/Lee/40 virus (B-Lee), orSendai virus (Sendai). B-Lee infected eggs were incubated at 35° C. foradditional 40 h. All other eggs were incubated at 37° C. for additional40 h. Virus present in the allantoic fluid was titrated byhemagglutination assay.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetically engineered attenuatedviruses and methods for their production. In particular, the presentinvention relates to engineering live attenuated viruses which contain amodified NS gene segment. Recombinant DNA techniques can be utilized toengineer site specific mutations into one or more noncoding regions ofthe viral genome which result in the down-regulation of one or moreviral genes. Alternatively, recombinant DNA techniques can be used toengineer a mutation, including but not limited to an insertion,deletion, or substitution of an amino acid residue(s) or an epitope(s)into a coding region of the viral genome so that altered or chimericviral proteins are expressed by the engineered virus.

The present invention further relates to a novel selection system toidentify influenza viruses containing a modified NS gene segment. Theselection system of the present invention is based, in part, on thehost-restriction of wild-type influenza virus and the ability ofinfluenza virus carrying a modification in the NS gene segment to infectand grow in an IFN-deficient cell.

The present invention is based, in part, on the Applicants' surprisingdiscovery that an engineered influenza A virus deleted of the NS1 genesegment is able to grow in a cell line deficient in IFN production, butis undetectable in Madin-Darby canine Kidney (MDCK) cells and in theallantoic membrane of embryonated chicken eggs, two conventionalsubstrates for influenza virus. The engineered influenza virus deletedof NS1 was further found by Applicants to induce IFN responses in humancells. The Applicants also found that an engineered influenza virusesdeleted of NS1 is capable of replicating and inducing disease in animalsthat were deficient in IFN signaling, but is nonpathogenic in wild-typemice.

The present invention further relates to the use of the attenuatedviruses of the present invention as a vaccine against a broad range ofviruses and/or antigens, including tumor specific antigens. Many methodsmay be used to introduce the live attenuated virus formulations to ahuman or animal subject to an immune response. These include, but arenot limited to, oral, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous and intranasal routes. In a preferredembodiment, the attenuated viruses of the present invention areformulated for delivery as an intranasal vaccine.

5.1. Attenuated Viruses which Induce Interferon Responses

The present invention relates to genetically engineered negative strandRNA viruses containing a modification, mutation, substitution, ordeletion in the gene whose product is responsible for the virus bypassof the cellular interferon response. Thus, the present invention relatesto genetically engineered RNA viruses, both segmented and non-segmented,containing a mutation in the gene responsible for down-regulating thecellular IFN response. The genetically engineered attenuated viruses ofthe present invention have an interferon-inducing phenotype, as opposedto the wild-type viruses which inhibit cellular interferon mediatedresponses.

In a preferred embodiment, the present invention relates to attenuatedinfluenza viruses with a modified NS gene segment and methods ofidentifying those modified influenza viruses. The present invention isbased, in part, on the discovery that although a NS1 modified virus isable to grow in IFN deficient cells, such as, Vero cells, its ability toreplicate was severely impaired in MDCK cells and in embryonated chickeneggs. It could be possible that these growth deficiencies are due tochanges in RNA segments other than the NS gene. In order to rule outthis possibility, the virus was “repaired” by rescuing an engineeredwild-type NS gene into the delNS1 virus. The resulting transfectantvirus grew to wild-type levels in MDCK cells and in eggs, demonstratingthat the lack of the NS1 gene determines the phenotypic characteristicsof the delNS1 virus.

Since NS modified viruses are capable of replicating in Vero cells whichare deficient in IFN expression, this indicates that altered tissueculture and egg growth of NS modified viruses is due to IFN-mediatedeffects. The following evidence supports the role of IFN-mediatedeffects: (a) The levels of viral protein expression are similar indelNS1 virus-infected Vero and in IFNαR−/− cells, but that they aremarkedly reduced in MDCK cells. It should be noted that IFNαR−/− cellsand Vero cells are both deficient in inducing an antiviral IFN response,although the genetic defect responsible for this deficiency is differentfor these two cell lines. (b) Infection with the NS modified virus butnot with wild-type virus induced transactivation of an IFN-stimulatedreporter gene in 293 cells. (c) Finally, the delNS1 virus was able toreplicate and to induce disease in mice that were deficient in IFNsignaling, i.e. STAT1−/− animals, but the virus was nonpathogenic inwild-type mice.

The importance of type I IFN is illustrated by the fact that manyviruses express antagonists which counteract IFN-mediated responses bythe host. Examples include VA RNAs of adenoviruses, the Epstein-Barrvirus-encoded structural small RNAs, the K3L and E3L gene products ofvaccinia virus, the NSP3 gene product of group C rotavirus, and thereovirus σ3 protein, among others. It is interesting that several ofthese viral products, like the NS1 protein of influenza A virus, areable to bind to dsRNA preventing activation of PKR. Thus, the attenuatedviruses of the present invention may also be used to supplement anyanti-viral therapeutic in that it enhances the IFN-mediated response, aresponse that most viruses have developed complex mechanisms to bypass.

The attenuated influenza virus of the present invention may be used toexpress heterologous sequences, including viral and tumor antigens.Thus, the attenuated viruses may be used to express a wide variety ofantigenic epitopes, i.e., epitopes that induce a protective immuneresponse to any of a variety of pathogens, or antigens that bindneutralizing antibodies may be expressed by or as part of the chimericviruses. The attenuated virus of the present invention is an excellentvehicle to introduce antigenic epitopes given that it induces anIFN-mediated response and it is not pathogenic to the host.

In accordance with the present invention, the genetic manipulation ofthe NS gene of influenza A viruses may help in generating viral vaccinevectors which express novel antigens and/or polypeptides. Since the NSRNA segment is the shortest among the eight viral RNAs, it is possiblethat the NS RNA will tolerate longer insertions of heterologoussequences than other viral RNAs. Moreover, the NS RNA segment directsthe synthesis of high levels of protein in infected cells, suggestingthat it would be an ideal segment for insertions of foreign antigens.However, in accordance with the present invention any one of the eightsegments of influenza may be used for the insertion of heterologoussequences.

Heterologous gene coding sequences flanked by the complement of theviral polymerase binding site/promoter, e.g, the complement of3′-influenza virus terminus, or the complements of both the 3′- and5′-influenza virus termini may be constructed using techniques known inthe art. Recombinant DNA molecules containing these hybrid sequences canbe cloned and transcribed by a DNA-directed RNA polymerase, such asbacteriophage T7, T3 or the Sp6 polymerase and the like, to produce therecombinant RNA templates which possess the appropriate viral sequencesthat allow for viral polymerase recognition and activity.

One approach for constructing these hybrid molecules is to insert theheterologous coding sequence into a DNA complement of an influenza virusgenomic segment so that the heterologous sequence is flanked by theviral sequences required for viral polymerase activity; i.e., the viralpolymerase binding site/promoter, hereinafter referred to as the viralpolymerase binding site. In an alternative approach, oligonucleotidesencoding the viral polymerase binding site, e.g., the complement of the3′-terminus or both termini of the virus genomic segments can be ligatedto the heterologous coding sequence to construct the hybrid molecule.The placement of a foreign gene or segment of a foreign gene within atarget sequence was formerly dictated by the presence of appropriaterestriction enzyme sites within the target sequence. However, recentadvances in molecular biology have lessened this problem greatly.Restriction enzyme sites can readily be placed anywhere within a targetsequence through the use of site-directed mutagenesis (e.g., see, forexample, the techniques described by Kunkel, 1985, Proc. Natl. Acad.Sci. U.S.A. 82;488). Variations in polymerase chain reaction (PCR)technology, described infra, also allow for the specific insertion ofsequences (i.e., restriction enzyme sites) and allow for the facileconstruction of hybrid molecules. Alternatively, PCR reactions could beused to prepare recombinant templates without the need of cloning. Forexample, PCR reactions could be used to prepare double-stranded DNAmolecules containing a DNA-directed RNA polymerase promoter (e.,bacteriophase T3, T7 or Sp6) and the hybrid sequence containing theheterologous gene and the influenza viral polymerase binding site. RNAtemplates could then be transcribed directly from this recombinant DNA.In yet another embodiment, the recombinant RNA templates may be preparedby ligating RNAs specifying the negative polarity of the heterologousgene and the viral polymerase binding site using an RNA ligase. Sequencerequirements for viral polymerase activity and constructs which may beused in accordance with the invention are described in the subsectionsbelow.

5.2. Generation of Attenuated Viruses

The present invention relates to genetically engineered attenuatedviruses, and methods for their production. In particular, the inventionrelates to attenuated influenza viruses which have been modified in sucha way to result in an IFN-independent and IFN-inducing phenotype. Thefollowing section describes the various approaches which may be used inaccordance with the invention to generate an attenuated phenotype.Recombinant DNA techniques can be utilized to engineer site specificmutations into one or more noncoding regions of the viral genome whichresult in the down-regulation of one or more viral gene. Alternatively,recombinant DNA techniques can be used to engineer a mutation, includingbut not limited to an insertion, deletion, or substitution of an aminoacid residue(s) or an epitope(s) into a coding region of the viralgenome so that altered or chimeric viral proteins are expressed by theengineered virus. The invention is based, in part, on the discovery thatthe down regulation of a viral gene in segmented viruses results in theproduction of defective particles at each round of replication, so thatthe virus demonstrates attenuated characteristics. In non-segmentedviruses, the down-regulation of a viral gene may result in theproduction of fewer progeny virions than would be generated by thecorresponding wild type virus. The alterations of the viral proteinsdescribed also result in attenuation for reasons which are less wellunderstood.

Many methods may be used to introduce the live attenuated virusformulations to a human or animal subject to induce an immune response;these include, but are not limited to, oral, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous and intranasal routes. It ispreferable to introduce the chimeric virus vaccine via its natural routeof infection.

Any virus may be engineered in accordance with the invention to producean attenuated strain suitable for use as a safe live-virus vaccine,including but not limited to viruses belonging to the families set forthin Table I below.

TABLE I FAMILIES OF HUMAN AND ANIMAL VIRUSES VIRUS CHARACTERISTICS VIRUSFAMILY dsDNA Enveloped Poxviridae Irididoviridae HerpesviridaeNonenveloped Adenoviridae Papovaviridae Hepadnaviridae ssDNANonenveloped Parvoviridae dsRNA Nonenveloped Reoviridae BirnaviridaessRNA Enveloped Positive-Sense Genome No DNA Step in ReplicationTogaviridae Flaviviridae Coronaviridae Hepatitis C Virus DNA Step inReplication Retroviridae Negative-Sense Genome Non-Segmented GenomeParamyxoviridae Rhabdoviridae Filoviridae Segmented GenomeOrthomyxoviridae Bunyaviridae Arenaviridae Nonenveloped PicornaviridaeCalciviridae Abbreviations used: ds = double stranded; ss = singlestranded; enveloped = possessing an outer lipid bilayer derived from thehost cell membrane; positive-sense genome = for RNA viruses, genomesthat are composed of nucleotide sequences that are directly translatedon ribosomes, = for DNA viruses, genomes that are composed of nucleotidesequences that are the same as the mRNA; negative-sense genome = #genomes that are composed of nucleotide sequences complementary to thepositive-sense strand.

DNA viruses (e.g., vaccinia, adenoviruses, baculovirus) and positivestrand RNA viruses (e.g., poliovirus) may be readily engineered usingrecombinant DNA techniques which are well known in the art (e.g., seeU.S. Pat. No. 4,769,330 to Paoletti; U.S. Pat. No. 4,215,051 to Smith;Racaniello et al., 1981, Science 214: 916-919). Until recently, however,negative strand RNA viruses (e.g., influenza) were not amenable to sitespecific genetic manipulation because the viral RNAs are not infectious.However, a recently developed technique, called “reverse genetics,”allows the engineering and production of recombinant negative strand RNAviruses.

The reverse genetics technique involves the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative strand virus which are essential for the recognition of viralRNA by viral polymerases and for packaging signals necessary to generatea mature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during in vitro transcription ofthe synthetic RNAs. The synthetic recombinant RNPs can be rescued intoinfectious virus particles. The foregoing techniques are described inU.S. Pat. No. 5,166,057, issued Nov. 24, 1992 and in Enami & Palese,1991, J. Virol. 65: 2711-2713, each of which is incorporated byreference herein in its entirety), and influenza A viruses containinginsertions, deletions and mutations with the stalk portion of the NSgene, which changes acts as a host range mutant.

5.2.1. Down-Regulation of Viral Genes

In accordance with the invention, a non-coding regulatory region of avirus can be altered to down-regulate a viral gene involved indown-regulating the cellular IFN-mediated response, e.g. reducetranscription of its mRNA and/or reduce replication of vRNA (viral RNA),so that an attenuated virus with an IFN-inducing phenotype is produced.

This approach, while applicable to any virus, is particularly attractivefor engineering viruses with segmented genomes; i.e., viruses in whichthe genome is divided into segments that are packaged into virions. Forexample, the segmented genome of influenza A virus (an orthomyxovirus)consists of eight molecules of linear negative-sense ssRNAs which encodeten polypeptides, including: the RNA-directed RNA polymerase proteins(PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid;two surface glycoproteins which project from the envelope: hemagglutinin(HA) and neuraminidase (NA); and nonstructural proteins (NS1 and NS2)whose function is unknown. The termini of each segment contain thenon-coding regions essential for recognition by viral polymerase and forpackaging signals necessary to generate a mature virion. The sequence ofthe termini is highly conserved among all eight segments. As anotherexample, the segmented genome of reoviruses consists of 10 to 12segments of linear dsRNA which encode 6 to 10 major structuralpolypeptides, a transcriptase and other enzymes.

The foregoing approach is equally applicable to non-segmented RNAviruses, where the down regulation of transcription of a viral geneinvolved in down-regulating the cellular IFN-mediated response, suchthat it will reduce the production of its mRNA and the encoded geneproduct and result in an interferon-inducing phenotype.

Any alteration of the regulatory non-coding regions which decrease theirefficiency or strength may be engineered in accordance in the invention.For example, the strength of viral promoters can be reduced byalterations in the stem structure. In order to achieve an attenuatedphenotype the cis elements of a virus gene involved in down-regulatingthe cellular IFN-mediated response may be mutated to achieve a dramaticeffect on transcription and replication of the gene.

How influenza A virus packages its eight RNA genome segments remains aninteresting question. In the past, two different mechanisms wereproposed for the packaging of influenza virus RNAs: one suggests thatthe eight RNAs are selectively packaged and the other that viral RNAsare packaged randomly (Compans et al., 1970, In The Biology Of Large RNAViruses, Barry & Mahy, Eds., pp. 87-108, Academic Press, N.Y.; Lamb &Choppin, 1983, Ann. Rev. Biochem. 467-506; Smith & Hay, 1982, Virology118: 96-108). Evidence is now accumulating to support the randompackaging mechanism. The random packaging theory originated from thefact that influenza viruses have a low ratio of infectious particles tophysical particles. If one assumes that an average of 11 RNAs arepackaged per virion, the expected ratio is compatible with that found invivo (Enami et al., 1991, Virology 185: 291-298). This model was alsosupported by the finding of a reassortant virus which contained twocopies of the same segment derived from two different viruses(Scholtissek, 1978, Virology 89: 506-516), and further support for thistheory came from a more recent report which described an influenza Avirus which required nine RNAs in order to be infectious (Enami et al.,1991, Virology 185: 291-298).

In summary, an attenuated phenotype may be achieved by targeting the ciselements of the NS gene segment to result in down regulation of the genesegment. Since the proteins of this virus are unaltered as compared towild type virus, attenuation must be the result of inefficientcis-acting signals. This principal of attenuation may be appliedanalogously to other viruses with segmented genomes. For example, theintroduction of modifications into the noncoding sequences of rotavirusgenes or of genes of other segmented dsRNA viruses (Roner et al., 1990,Virology 179: 845-852) should also allow the pathogenicity of theseviruses to be altered.

5.2.2. Alteration of Viral Proteins

An alternative way to engineer attenuated viruses involves theintroduction of an alteration, including but not limited to aninsertion, deletion or substitution of one or more amino acid residuesand/or epitopes into one or more of the viral proteins involved indown-regulating the cellular IFN-mediated response. This may be readilyaccomplished by engineering the appropriate alteration into thecorresponding viral gene sequence. Any change that alters the activityof the viral protein involved in down-regulating the cellularIFN-mediated response so that viral replication is modified or reducedmay be accomplished in accordance with the invention.

For example, alterations that interfere with but do not completelyabolish viral attachment to host cell receptors and ensuing infectioncan be engineered into viral surface antigens or viral proteasesinvolved in processing to produce an attenuated strain. According tothis embodiment, viral surface antigens can be modified to containinsertions, substitution or deletions of one or more amino acids orepitopes that interfere with or reduce the binding affinity of the viralantigen for the host cell receptors. This approach offers an addedadvantage in that a chimeric virus which expresses a foreign epitope maybe produced which also demonstrates attenuated characteristics. Suchviruses are ideal candidates for use as live recombinant vaccines. Forexample, heterologous gene sequences that can be engineered into thechimeric viruses of the invention include, but are not limited to,epitopes of human immunodeficiency virus (HIV) such as gp120; hepatitisB virus surface antigen (HBsAg); the glycoproteins of herpes virus(e.g., gD, gE); VP1 of poliovirus; and antigenic determinants ofnonviral pathogens such as bacteria and parasites to name but a few.

In this regard, influenza is an ideal system in which to engineerforeign epitopes, because the ability to select from thousands ofinfluenza virus variants for constructing chimeric viruses obviates theproblem of host resistance or immune tolerance encountered when usingother virus vectors such as vaccinia. In addition, since influenzastimulates a vigorous secretory and cytotoxic T cell response, thepresentation of foreign epitopes in the influenza background may alsoprovide for the secretory immunity and cell-mediated immunity. By way ofexample, the insertion, deletion or substitution of amino acid residuesin the HA protein of influenza can be engineered to produce anattenuated strain. In this regard, alterations to the B region or Eregion of HA may be utilized. In accordance with this approach, themalarial epitope (ME 1) of Plasmodium yoelii (NEDSYVPSAEQI; SEQ ID NO:5)was introduced into the antigenic site E of the hemagglutinin ofinfluenza. The resulting chimeric virus has a 500- to 1,000-fold lowerLD₅₀ (lethal dose 50) than that of wild type virus when assayed in mice.In another embodiment, the major antigenic determinant of poliovirustype 1, i.e., the BC loop of the VP1 of poliovirus type 1 (PASTTNKDKL;SEQ ID NO:6) was engineered into the B region of the influenza HAprotein. This chimeric virus is also attenuated.

In another embodiment, alterations of viral proteases required forprocessing viral proteins can be engineered to produce attenuation.Alterations which affect enzyme activity and render the enzyme lessefficient in processing, should affect viral infectivity, packaging,and/or release to produce an attenuated virus. For example, alterationsto the NS protein of influenza can be engineered to reduce NS enzymeactivity and decrease the number and/or infectivity of progeny virusreleased during replication.

In another embodiment, viral enzymes involved in viral replication andtranscription of viral genes, e.g., viral polymerases, replicases,helicases, etc. may be altered so that the enzyme is less efficient oractive. Reduction in such enzyme activity may result in the productionof fewer progeny genomes and/or viral transcripts so that fewerinfectious particles are produced during replication.

The alterations engineered into any of the viral enzymes include but arenot limited to insertions, deletions and substitutions in the amino acidsequence of the active site of the molecule. For example, the bindingsite of the enzyme could be altered so that its binding affinity forsubstrate is reduced, and as a result, the enzyme is less specificand/or efficient. For example, a target of choice is the viralpolymerase complex since temperature sensitive mutations exist in allpolymerase proteins. Thus, changes introduced into the amino acidpositions associated with such temperature sensitivity can be engineeredinto the viral polymerase gene so that an attenuated strain is produced.

5.3. Host-Restriction Based Selection System

The present invention relates to a host-restriction based selectionsystem for the identification of genetically manipulated influenzaviruses. The selection system of the present invention is moreparticularly related to the identification of genetically manipulatedinfluenza viruses which contain modified NS gene segments. The selectionsystem of the present invention allows for the screening of thegenetically engineered influenza viruses to identify those viruses witha modified NS gene segment.

The selection system of the present invention is based, in part, on theApplicants' discovery that an engineered influenza A virus deleted ofthe NS1 gene was able to grow in a cell line deficient in IFNproduction, whereas the same cell line would not support infection andgrowth of wild type influenza virus. The NS1 deleted virus was unable toinfect and grow in the conventional substrates for influenza virus.Thus, the invention provides a very simple and easy screen to identifythose genetically engineered influenza viruses that contain a modifiedNS1 gene.

5.4. Vaccine Formulations Using the Chimeric Viruses

Virtually any heterologous gene sequence may be constructed into thechimeric viruses of the invention for use in vaccines. Preferably,epitopes that induce a protective immune response to any of a variety ofpathogens, or antigens that bind neutralizing antibodies may beexpressed by or as part of the chimeric viruses. For example,heterologous gene sequences that can be constructed into the chimericviruses of the invention for use in vaccines include but are not limitedto epitopes of human immunodeficiency virus (HIV) such as gp120;hepatitis B virus surface antigen (HBsAg); the glycoproteins of herpesvirus (e.g. gD, gE); VP1 of poliovirus; antigenic determinants ofnon-viral pathogens such as bacteria and parasites, to name but a few.In another embodiment, all or portions of immunoglobulin genes may beexpressed. For example, variable regions of anti-idiotypicimmunoglobulins that mimic such epitopes may be constructed into thechimeric viruses of the invention.

Either a live recombinant viral vaccine or an inactivated recombinantviral vaccine can be formulated. A live vaccine may be preferred becausemultiplication in the host leads to a prolonged stimulus of similar kindand magnitude to that occurring in natural infections, and therefore,confers substantial, long-lasting immunity. Production of such liverecombinant virus vaccine formulations may be accomplished usingconventional methods involving propagation of the virus in cell cultureor in the allantois of the chick embryo followed by purification.

In this regard, the use of genetically engineered influenza virus(vectors) for vaccine purposes may require the presence of attenuationcharacteristics in these strains. Current live virus vaccine candidatesfor use in humans are either cold adapted, temperature sensitive, orpassaged so that they derive several (six) genes from avian viruses,which results in attenuation. The introduction of appropriate mutations(e.g., deletions) into the templates used for transfection may providethe novel viruses with attenuation characteristics. For example,specific missense mutations which are associated with temperaturesensitivity or cold adaption can be made into deletion mutations. Thesemutations should be more stable than the point mutations associated withcold or temperature sensitive mutants and reversion frequencies shouldbe extremely low.

Alternatively, chimeric viruses with “suicide” characteristics may beconstructed. Such viruses would go through only one or a few rounds ofreplication in the host. For example, cleavage of the HA is necessary toallow for reinitiation of replication. Therefore, changes in the HAcleavage site may produce a virus that replicates in an appropriate cellsystem but not in the human host. When used as a vaccine, therecombinant virus would go through a single replication cycle and inducea sufficient level of immune response but it would not go further in thehuman host and cause disease. Recombinant viruses lacking one or more ofthe essential influenza virus genes would not be able to undergosuccessive rounds of replication. Such defective viruses can be producedby co-transfecting reconstituted RNPs lacking a specific gene(s) intocell lines which permanently express this gene(s). Viruses lacking anessential gene(s) will be replicated in these cell lines but whenadministered to the human host will not be able to complete a round ofreplication. Such preparations may transcribe and translate—in thisabortive cycle—a sufficient number of genes to induce an immuneresponse. Alternatively, larger quantities of the strains could beadministered, so that these preparations serve as inactivated (killed)virus vaccines. For inactivated vaccines, it is preferred that theheterologous gene product be expressed as a viral component, so that thegene product is associated with the virion. The advantage of suchpreparations is that they contain native proteins and do not undergoinactivation by treatment with formalin or other agents used in themanufacturing of killed virus vaccines.

In another embodiment of this aspect of the invention, inactivatedvaccine formulations may be prepared using conventional techniques to“kill” the chimeric viruses. Inactivated vaccines are “dead” in thesense that their infectivity has been destroyed. Ideally, theinfectivity of the virus is destroyed without affecting itsimmunogenicity. In order to prepare inactivated vaccines, the chimericvirus may be grown in cell culture or in the allantois of the chickembryo, purified by zonal ultracentrifugation, inactivated byformaldehyde or β-propiolactone, and pooled. The resulting vaccine isusually inoculated intramuscularly.

Inactivated viruses may be formulated with a suitable adjuvant in orderto enhance the immunological response. Such adjuvants may include butare not limited to mineral gels, e.g., aluminum hydroxide; surfaceactive substances such as lysolecithin, pluronic polyols, polyanions;peptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum.

Many methods may be used to introduce the vaccine formulations describedabove, these include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. It may be preferable to introduce the chimeric virusvaccine formulation via the natural route of infection of the pathogenfor which the vaccine is designed. Where a live chimeric virus vaccinepreparation is used, it may be preferable to introduce the formulationvia the natural route of infection for influenza virus. The ability ofinfluenza virus to induce a vigorous secretory and cellular immuneresponse can be used advantageously. For example, infection of therespiratory tract by chimeric influenza viruses may induce a strongsecretory immune response, for example in the urogenital system, withconcomitant protection against a particular disease causing agent.

6. Materials and Methods

The following materials and methods were used in the following Sections7 through 11.

Viruses and cells. Influenza A/PR/8/34 (PR8) virus was propagated in10-day-oldembryonated chicken eggs at 37° C. Influenza A virus 25A-1, areassortant virus containing the NS segment form the cold-adapted strainA/Leningrad/134/47/57 and the remaining genes from PR8 virus (Egorov etal., 1994, Vopr. Virusol. 39:201-205; Shaw et al., 1996, in Options forthe control of influenza III, eds. Brown, Hampson Webster (ElsevierScience) pp. 433-436) was grown in Vero cells at 34° C. The 25A-1 virusis ts in mammalian cells, and was used as helper virus for the rescue ofthe delNS1 transfectant virus. Vero cells and MDCK cells in minimalessential medium (MEM) containing 1 μg/ml of trypsin (DifcoLaboratories, Detroit, Mich.) were used for influenza virus growth. Verocells were also used for selection, plaque purification and titration ofthe delNS1 virus. MDCK cells, 293 cells and mouse embryo fibroblasts(MEF) derived from 14-16 day embryos of IFNαR−/− mice were maintained inDMEM (Dulbecco's minimal essential medium) containing 10%heat-inactivated detal calf serum. Immortalized IFNαR−/− fibroblastswere derived from MEF by continuous passage (Todaro et al., 1963, J.Cell. Biol. 17:299-313). Vero cells were grown in AIM-V medium (LifeTechnologies, Grand Island, N.Y.).

Mice. C57BL/6 mice homozygous for a targeted deletion of STAT1 weregenerated as previously described (Durbin et al., 1996, Cell84:443-450). IFNαR−/− mice have also been described (Hwang et al., 1995,Proc. Natl. Aced. Sci. USA 92:11284-11288). Specific pathogen freeC57BL/6 and BALB/c (wild type) mice were purchased from Taconic Farms.

Animal infections. Female mice were used for influenza virus infectionat 6 to 12 weeks of age. Intranasal (i.n.) inoculations were performedin wild type and STAT1−/− mice under ether anesthesia using 50 μl of MEMcontaining 5×10⁴ plaque forming units (pfu) of delNS1 virus. Animalswere monitored daily, and sacrificed when observed in extremis. Allprocedures were in accord with NIH guidelines on care and use oflaboratory animals.

Plasmids. pT3delNS1 was made as follows.

First, pPUC19-T3/NS PR8, containing the complete NS gene of PR8 virusflanked by the T3 RNA polymerase promoter and BpuAI restriction site wasamplified by inverse PCR (Ochman et al., 1988, Genetics 120:621-623)using primers 5′-CTGAAAGCTTGACACAGTGTTTG-3′ (SEQ ID NO:1) and5-GACATACTGCTGAGGATGTC-3′ (SEQ ID NO:2) (CODON Genetic Systems, Weiden,Austria). The obtained cDNA thus lacking the NS1 gene wasphosphorylated, Klenow treated, self-ligated and propagated in E. colistrain TG1. The construct obtained alter purification was namedpT3delNS1 and verified by sequencing. Plasmids for expression of the NP,PB1, PB2, and PA proteins of PR8 virus (pHMG-NP, pHMG-PB1, pHMG-PB2, andpHMG-PA) were previously described (Pleschka et al., 1996, J. Virol.70:4188-4192). pPOLI-NS-RB was made by substituting the CAT open readingframe of pPOLI-CAT-RT (Pleschka et al., 1996, J. Virol. 70:4188-4192)within RT-PCR product derived from the coding region of the NS gene ofinfluenza A/WSN/33 (WSN) virus. This plasmid expresses the NS-specificviral RNA segment of WSN virus under the control of a truncated humanpolymerase I promoter. pHISG54-1-CAT (Bluyssen et al., 1994, Eur. J.Biochem. 220:395-402) encodes the CAT reporter gene under thetranscriptional control of the IFNα-stimulated promoter of the ISG-54Kgene.

Generation of transfectant viruses. Generation of delNS1 virus wasperformed by ribonucleoprotein (RNP) transfection (Luytijes et al.,1989, Cell 59:1107-1113). The RNPs were formed by T3 RNA polymerasetranscription from pT3delNS1 linearized with BpuAI in the presence ofpurified nucleoprotein and polymerase of influenza 25A-1 virus (Enami etal., 1991, J. Virol 65: 2711-2713). RNP complexes were transfected intoVera cells which were previously infected with 25A-1 virus. Transfectedcells were incubated for 18 hours at 37° C., and the supernatant waspassaged twice in Vero cells at 40° C. and plague purified three timesin Vera cells covered with agar overlay media at 37° C. The isolateddelNS1 virus was analyzed by RT-PCR using primers5′GGCCTCTAGATAATACGACTC-ACTATAAGCAAAAGCAGGGTGACAAAG-3′ (SEQ ID NO:3)(complementary to position 1 to 21 at the 3′ noncoding end of the NSgene) and 5 ′-GATCGCCTTCTATTAGTAGAAA-CAAGGGTGTTTTTATTAAATAAGCTG-3′ (SEQID NO:4) (containing the last 38 nucleotides of the 5′ noncoding end ofthe NS gene). NS/WSN transfectant virus was generated as follows. Veracells in 35-mm dishes were transfected with plasmids pHMG-NP, pHMG-PB1,pHMG-PB2, pHMG-PA and pPOLI-NS-RB, as previously described (Pleschka etal., 1996, J. Virol. 70:4188-4192). 2 days postransfection, cells wereinfected with 5×10⁴ pfu of delNS1 virus and incubated 2 more days at 37°C. Cell supernatant was passaged once in MDCK cells and twice in chickenembryonated eggs. Transfectant viruses were cloned by limiting dilutionin eggs. Genomic RNA from purified NS/WSN transfectant virus wasanalyzed by polyacrylamide gel electrophoresis, as previously described(Zheng et al., 1996, Virology 217:242-251).

Analysis of virus protein synthesis in infected cells. Cell monolayersin 35-mm dishes were infected with 2×10⁴ pfu of delNS1 virus. Atintervals postinfection, cells were labeled with L-[³⁵S]cysteine andL-[³⁵S] methionine for the indicated times. Labeled cells were lysed in10 mM tris-HCl (pH 7.4) containing 150 mM NaCl, 5 mM EDTA, 1 mM PMSF,10% glycerol, 1% Triton X-100, 1% sodium deoxycholate and 0.1% sodiumdodecylsufate (SDS). Proteins were immunoprecipitated using a rabbitpolyclonal anti-influenza virus serum. Immunoprecipated proteins wereanalyzed by SDS-10% polyacrylamide gel electrophoresis (SDS-PAGE).

CAT transfections. 293 cell monolayers in 35-mm dishes were transfectedwith 1 μg of pHISG54-1-CAT using DOTAP lipofection reagent (BoehringerMannheim) according to the manufacturer's instructions, and incubated at37° C. 1 day postransfection cells were infected with delNS1 virus orPR8 virus at the indicated multiplicities of infection (MOI). Ascontrols, cells were mock-infected or transfected with 50 μg ofpoly(I-C). After 1 day more at 37° C., cell extracts were made andassayed for CAT activity, as described (Percy et al., 1994, J. Virol.68:4486-4492).

7. EXAMPLE Generation of the Transfectant Influenza Virus delNS1,Lacking the NS1 Gene

The NS-specific viral RNA segment of influenza A virus encodes both theNS1 and NEP (nuclear export protein) proteins. Unspliced NS-specificmRNA translates into the NS1 protein, while the spliced RNA directs thesynthesis of the NEP. The plasmid pT3delNS1 was constructed whichexpresses a mutated NS gene from influenza PR8 virus. This mutated RNAsegment contains a deletion of the NS1-specific open reading frame (ntpositions 57 to 528 of the PR8 NS gene (Baez et al., 1980, Nucleic AcidsRes. 8:5845-5858) and thus it encodes only the NEP (FIG. 1). RNPtransfection of the delNS1 gene using the ts 25A-1 helper virus yieldeda progeny virus which was able to grow at 40° C. in Vero cells.Amplification of the NS gene of the rescued virus by RT-PCR confirmedthe substitution of the NS gene of the helper virus with that derivedfrom the transfected delNS1 gene (FIG. 2).

8. EXAMPLE Growth Properties of delNS1 Virus in Tissue Culture and Eggs

The growth properties of delNS1 virus and wild-type PR8 virus werecompared in Vero cells, MDCK cells, and 10-day-old embryonated chickeneggs. Cell monolayers containing approximately 10⁶ Vero or MDCK cellswere infected with delNS1 virus or PR8 virus at an MOI of approximately0.0005. After 4 days incubation at 37° C. using MEM containing 1 μg/mlof trypsin, supernatants were used in a hemagglutination assay.Alternatively, the allantoic cavity of 10-day-old embryonated chickeneggs was injected with 10⁴ pfu of delNS1 or PR8 virus, and thehemagglutination titer in the allantoic fluids was determined after 3days of incubation of 37° C. As shown in Table 2, delNS1 virus was ableto grow in Vero cells to titers of 16 as compared to a tier of 128 forwild-type PR8 virus. However, delNS1 virus replication was severelyimpaired in MDCK cells and in eggs (hemagglutination titers wereundetectable in these samples).

TABLE 2 DelNS1 virus replication in tissue culture cells and eggsHemagglutination titer¹ Culture media delNS1 WT PR8² Vero cells 16 128MDCK cells <2 512 Eggs <2 2,048 ¹Titers represent the highest dilutionwith hemagglutinating activity ²Wild type influenza A/PR/8/34 virus

8.1. Deletion of the NS1 Gene is Responsible for the Growth Propertiesof delNS1

In order to prove that the impaired viral growth of delNS1 virus in MDCKcells and eggs was due to the deletion of the NS1 gene and not topossible differences in other RNA segments of delNS1 and PR8 viruses, weused delNS1 virus as helper virus to rescue a wild-type NS gene.Transfections were carried as described in Materials and Methods using aplasmid-based expression system (Pleschka et al., 1996, J. Virol.70:4188-4192) for the wild-type NS gene of influenza A/WSN/34 virus.Selection of transfectant NS/WSN viruses were done by serial passages inMDCK cells and eggs. SDS-PAGE analyses of purified viral RNA from NS/WSNvirus confirmed the wild-type length of its NS RNA segment. TransfectantNS/WSN virus containing the NS RNA segment derived from WSN virus andthe remaining segments from delNS1 (PR8) virus was able to grow toidentical titers than those of wild-type PR8 virus in MDCK cells and10-day-old embryonated chicken eggs.

8.2. Viral Protein Expression Levels in delNS1 Virus-infected MDCK andVero Cells

In order to investigate if the deficiency of delNS1 virus replicationMDCK cells correlated with a decrease in the expression levels of viralproteins, [³⁵S]-labeling experiments were performed. MDCK or Vero cellswere infected with delNS1 virus at an MOI of 0.02 and labeled withL-[³⁵S]cysteine and L-[³⁵S]methionine for the indicated times. Afterlabeling, viral proteins were immunoprecipitated from cell extracts andseparated by PAGE (FIG. 3). Quantitation of the ³⁵S signal indicatesthat approximately 20-fold less viral protein was synthesized byinfected MDCK cells.

8.3. DelNS1 Virus-infected Vero and IFNαR−/− Cells have Similar Levelsof Viral Protein Expression

The reason for the differences between Vero and MDCK cells in supportingdelNS1 virus replication and viral protein expression may relate to theinability of Vero cells to synthesize IFN (Desmyter et al., 1968, J.Virol. 2:955-961; Mosca et al., 1986, Mol. Cell. Biol. 6:2279-2283; Diazet al., 1988, Proc. NeH. Acad. Sci. USA 85:52595263). In order to testthis hypothesis, the pattern of viral protein expression wasinvestigated in a murine cell line which is unable to respond to IFN.IFNαR knock-out cells were infected with delNS1 virus at an MOI of 0.02and labeled as described above. As a control, delNS1 virus-infected Verocells were labeled from 8 to 10 hours postinfection at the same time.Levels of viral protein expression were similar in both cell lines,suggesting that delNS1 virus is able to replicate in IFN-deficientsystems. It should be noted that we could not investigate multicyclereplication of delNS1 virus in IFNαR−/− cells because these cells dierapidly in the presence of trypsin, which is required for viralhemagglutinin activation and virus infectivity.

9. EXAMPLE Delta NS1 Virus is a Potent Inducer of Interferon Responses

Stimulation of transcription from an IFN-regulated promoter by infectionwith transfectant delNS virus. In order to investigate if the deletionof the NS gene in delNS virus results in an enhanced IFN response ininfected cells we performed transfection experiments using pHISG54-CAT.This plasmid contains the CAT reporter gene in front of theIFNα-stimulated promoter of the ISG-54K gene (Bluyssen et al., 1994,Eur. J. Biochem. 220:395-402). 293 cells were transfected withpHISG54-1-CAT and infected with delNS1 virus or wild-type PR8 at theindicated MOIs as described in Material and Methods. CAT activity in theinfected cell extracts was compared with that in uninfected cellextracts. As positive control, transcription from the IFN-regulatedpromoter was stimulated using poly (I-C). As shown in FIG. 5, infectionat an MOI of 0.05 with delNS1 virus, but not with wild-type virus,induced approximately a 6-fold stimulation of reporter gene expression.The transfectant virus lacking the NS1 gene was thus impaired in itsability to inhibit the IFN response in 293 infected cells.

10. EXAMPLE Pathogenicity of Delta NS1 Virus

Transfectant delNS1 virus is pathogenic in STAT1−/− mice. The ability ofdelNS1 virus to replicate and cause disease in wild-type mice and inmice deficient in the IFN responses was investigated. For this purpose,5×10⁴ pfu of delNS1 virus was used to i.n. infect three C57BL/6 mutantmice which were homozygous for a targeted deletion of STAT1, atransactivator which is required for the IFN signaling (Durbin et al.,1996, Cell 84:443-450; Merez et al., 1996, Cell84:431-442). Three tofour wild-type C57BL/6 and BALB/c mice were also inoculated with delNS1virus. Infected-STAT1−/− mice looked sick by day 3 postinfection. By day7 postinfection, all three infected STAT1−/− mice died (Table 3). DelNS1virus was recovered from the lungs of STAT1−/− dying mice, indicatingthat the virus was replicating in these animals. However, all wild-typeinfected mice survived infection with delNS1 without developing anysymptoms of disease (Table 3).

TABLE 3 Survival of mice following delNS1 virus infection¹ Daypostinfection Mice 1 day 7 day 14 day STAT1−/− C57BL/6 3 of 3 0 of 3 0of 3 Wild-type C57BL/6 3 of 3 3 of 3 3 of 3 Wild-type BALB/c 4 of 4 4 of4 4 of 4 ¹Mice under ether anesthesia were inoculated intranasally with5 × 10⁴ pfu of delNS1 virus

11. EXAMPLE Preinoculation with delNS1 Virus Inhibits Replication ofInfluenza

In order to investigate if preinoculation with the delNS1 virus has aninhibitory effect on infection or replication of wild-type influenza,the following experiment was conducted.

In this study 10-day old embroyated chicken eggs were inoculated with20,000 pfu of the delNS1 virus or with PBS into the allantoic cavity.After 8 hours of incubation at 37° C., the eggs were reinfected with 10³pfu of H1N1 influenza A/WSN/33 (WSN); H1N1 influenza A/PR/8 virus (PR8),H3N2 influenza A/X-31 virus (X-31), influenza B/Lee/40 virus (B-Lee) orSendai virus and incubated for an additional 40 hours at 37° C., exceptfor B-Lee infected cells which were incubated at 35° C. As shown in FIG.5, the delNS1 virus treated eggs resulted in undetectable levels ofviral infection, when compared to the untreated cells. Thus,demonstrating the anti-viral activity of the delNS1 virus and itspotential as an anti-viral therapeutic and prophylactic.

The present invention is not to be limited in scope by thespecific-embodiments described which are intended as singleillustrations of individual aspects of the invention, and any constructsor viruses which are functionally equivalent are within the scope ofthis invention. Indeed, various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An attenuated genetically engineered influenza A virus with aninterferon-inducing phenotype containing a knockout of the NS1 genesegment.
 2. An attenuated genetically engineered influenza A viruscontaining a deletion of the entire NS1 gene segment which is infectiousand replicates in an interferon (IFN) deficient cell line.
 3. Theattenuated virus of claim 1 or 2 which further encodes a heterologoussequence.
 4. The attenuated virus of claim 3 in which the heterologoussequence encodes a viral antigenic peptide.
 5. The attenuated virus ofclaim 3 in which the heterologous sequence encodes a tumor antigenicpeptide.
 6. A formulation comprising an attenuated geneticallyengineered influenza A virus containing a complete deletion in its NS1gene segment in a suitable pharmaceutical formulation.
 7. A formulationcomprising a genetically engineered attenuated chimeric influenza Avirus that expresses a heterologous sequence, said virus having aninterferon-inducing phenotype and a viral genome wherein the completeNS1 coding sequences have been deleted.
 8. The formulation of claim 7,wherein the heterologous sequence encodes a viral antigen.
 9. Theformulation of claim 8, wherein the viral antigen is humanimmunodeficiency virus gp120, hepatitis B virus surface antigen, aherpes virus glycoprotein, or VP1 of poliovirus.
 10. The formulation ofclaim 7, wherein the heterologous sequence encodes a tumor specificantigen.
 11. The formulation of claim 7, wherein the heterologoussequence encodes an immunoglobulin gene or a portion thereof.
 12. Theformulation of claim 7, wherein the heterologous sequence is themalarial epitope (ME 1) of Plasmodium yoelii.
 13. The formulation ofclaim 7 which is formulated for an intranasal administration.
 14. Amethod of inducing an immune response comprising administering to asubject the formulation of claim
 7. 15. A method of inducing an immuneresponse comprising administering to a subject the formulation of claim13.
 16. The method of claim 14, wherein the subject is an animal. 17.The method of claim 15, wherein the subject is an animal.
 18. The methodof claim 14, wherein the subject is a human.
 19. The method of claim 15,wherein the subject is a human.
 20. The method of claim 14, wherein theformulation is administered to the subject orally, intradermally,intramusclarly, intraperitoneally, intravenously, or subcutaneously. 21.The method of claim 15, wherein the formulation is administered to thesubject intranasally.
 22. The formulation of claim 7, wherein theheterologous sequence encodes a bacterial antigen.
 23. The formulationof claim 7, wherein the heterologous sequence encodes a parasiticantigen.